The hepatitis C virus (which may be abbreviated as “HCV”hereinafter) is an RNA virus that is classified as a member of the genus Hepacivirus of the family Flaviviridae. It has been identified as a major causative virus of non-A and non-B hepatitis (non-patent document 1). The HCV genome encodes a precursor protein that is converted into 10 types of virus protein (i.e., Core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) via post-translational cleavage by host-derived signal peptidase or HCV-derived proteases. Of these virus proteins, Core, E1, E2, and p7 proteins are classified as structural proteins, and NS2, NS3, NS4A, NS4B, NS5A, and NS5B proteins are classified as non-structural proteins.
HCV is mainly transmitted via blood transfusion. Highly sensitive methods for detecting HCV have been established today, and the number of new HCV patients because of blood transfusion has dramatically decreased. However, at present, the number of HCV carriers including so-called virus carriers who have not yet developed hepatitis symptoms is deduced to be over 2,000,000 in Japan, and is over 170,000,000 in the world. This is mainly because the rate of chronicity of hepatitis due to HCV infection is as high as 70% to 80% and there are no effective antiviral agents other than interferons at present. Further, chronic hepatitis C caused by HCV infection would become worse and lead to cirrhosis during the following some 20 years, finally resulting in liver cancer. Further, liver cancer is known to result in relapse for many patients due to inflammation that continuously occurs at noncancerous parts even if cancer is surgically excised.
Therefore, development of antiviral drugs and vaccines with beneficial effects has been desired for the purpose of preventing virus carriers from developing the disease and eliminating viruses. For this purpose, detailed information about the HCV life cycle should be clarified, such as regarding the ways in which HCV invades, replicates, grows in host cells, and HCV affects host cells.
The HCV life cycle involves the series of cycles described below. First, HCV binds to a specific protein (virus receptor) on the cell surface and is incorporated by endocytosis into the host cell. Next, HCV genomic RNA is released into the host cytoplasm from viral particles (uncoating). Subsequently, HCV protein precursors encoded by the released HCV genomic RNA are translated. After each virus protein has been generated by processing, the HCV genomic RNA is replicated by RNA polymerase, which is one of the generated virus proteins. The thus replicated HCV genomic RNA is packaged by the Core protein and envelope proteins (E1 protein and E2 protein), which are structural proteins, so that new viral particles are formed. Finally, viral particles break the host cell membranes and are then released from the cells.
Therefore, it is important to develop a method for inhibiting at least one of the above steps in the process of HCV infection, in order to prevent HCV carriers from developing the disease and to eliminate the virus.
HCV envelope proteins are considered to play a key role in the binding of HCV to cell surfaces. Thus, research has been conducted for preparation of antibodies against envelope proteins in blood serum samples of HCV patients. However, the percentage of HCV patients exhibiting positive reactions with either the C100 antibody (the NS4-NS-5 antibody) or the anti-core antibody, both of them or an anti-envelope protein antibody was found to be approximately 10%. Since only about 10% of HCV patients are naturally cured with a neutralizing antibody (non-patent document 2), it is thought that as few as 1% of all patients who are thought to be cured by the anti-envelope protein antibody. This is thought to be due to the presence of a mechanism that inhibits or suppresses the production of antibodies against HCV envelope proteins (non-patent document 3)
Meanwhile, non-patent document 4 discloses that when one of the HCV envelope proteins, the E2 protein, is expressed in a mammal, the E2 protein specifically binds to CD81 existing on human cell surfaces. Based on the experimental result, isolation of an antibody that exhibits NOB (neutralization of binding) activity that inhibits the binding between the E2 protein and CD81 from a hepatitis C patient has been attempted. For example, through construction of an antibody gene library from the bone-marrow lymphocytes of a chronic hepatitis C patient affected by HCV of genotype 1a, followed by employment of a phage display method, the above antibody has been isolated (patent document 1). Moreover, an antibody exhibiting NOB activity has also been isolated by a method for preparing hybridomas from peripheral B cells of a hepatitis C patient affected by HCV of genotype 1b (non-patent document 5 and patent document 2). However, with methods for preparing monoclonal antibodies from HCV patients, it is difficult to obtain a variety of repertoires of infection-inhibiting antibodies and to find antibodies useful as anti-HCV agents, since only the patients having HCV infection-inhibiting antibodies can be used herein. Also, it has been reported that an antibody exhibiting NOB activity does not always inhibit infection (non-patent document 8).
Furthermore, a method that involves inducing an antibody via administration of a recombinant envelope protein to a mouse (patent document 3) and a method that involves fusing lymphocytes to myeloma cells to prepare antibody-producing hybridomas (thus preparing an antibody against an envelope protein) have been attempted (patent document 4 and non-patent document 6). However, no effective antibody inhibiting HCV infection has been obtained to date. No antibody neutralizing HCV infection has been prepared by immunizing an animal with an envelope protein. One of the suggested reasons for this lack is that a recombinant envelope protein to be used for immunization has a structure differing from that of the virus's original envelope protein. It has also been reported that recombinant envelope proteins tend to aggregate so that they are unable to maintain their original conformations (non-patent document 7).
Therefore, in view of treatment and prevention using HCV antibodies, development of antibodies against envelope proteins that are capable of inhibiting viral infection and a new method for effectively inducing such antibodies have been desired.
Starting from the above background, technique for preparing infectious HCV particles with a cell culture system has been recently established (patent documents 5, 6, and 7). Unlike the above method, which involves causing the expression of a recombinant envelope protein by gene recombination techniques and using the resultant as an antigen, HCV particles prepared using such a cell culture system are infectious, and thus the conformation of the HCV antigen may be maintained.
The conformation of HCV is composed of an envelope comprising envelope proteins (E1 protein and E2 protein) and a lipid membrane. These E1 and E2 proteins are thought to bind to each other, forming a complex (non-patent document 7).
On the other hand, envelope proteins of an AIDS virus form a trimer. It has been revealed that an antibody recognizing the conformation of the trimer as an epitope (antigenic determinant) is effective against a wide range of AIDS viruses, compared with conventional anti-AIDS virus antibodies, and has high neutralization activity. This suggests that it is important for an antibody with such neutralization activity to be able to recognize the conformation of a viral antigen as an epitope (non-patent document 9).